Project Summary Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable and inherited mechano-bullous disease of the skin characterized by skin fragility, blister formation, and chronic wounds. RDEB is caused by mutations in the COL7A1 gene encoding type VII collagen (C7), the major component of anchoring fibrils (AFs) that anchor the epidermis to the dermis. AFs are attenuated, diminutive, or absent in RDEB. There is no effective treatment for RDEB except palliative measures and supportive wound care. Patients with RDEB die of an aggressive metastatic squamous cell carcinoma in the second or third decade of life. We used molecular and cellular approaches to study the structure and function of C7 and showed the feasibility of keratinocyte-based ex vivo gene therapy; fibroblast based-cell therapy; and recombinant C7-based protein therapy for potential treatment of RDEB. Approximately 25-30% of RDEB patients have nonsense mutations leading to premature stop codons (PTCs) and a truncated C7 with diminished function. Aminoglycoside antibiotics such as gentamicin are able to read-through these mutations and restore synthesis of a full-length functional protein. Using cultured skin cells from 4 RDEB patients with nonsense mutations, we recently showed that aminoglycosides were able to read-through PTCs and produce full- length C7. Importantly, aminoglycoside-induced C7 reversed the abnormal RDEB cell phenotype and incorporated into the dermal-epidermal junction (DEJ) of skin equivalents composed of cells with these mutations. We translated these in vitro findings in a recent pilot clinical trial study of 5 RDEB patients with nonsense mutations. We showed that intradermally injected and topical gentamicin readily induced PTC read-through and created robust new C7 and AFs at the patient's DEJ that were stable for months after treatment. Importantly, topical gentamicin also improved wound closure and skin quality. Further, we did not detect any gentamicin-associated side effects. Since RDEB patients have widespread lesions and involvement of the upper third of the esophagus, intravenous (IV) gentamicin has the potential of treating all skin and esophageal sites simultaneously. Based on our encouraging in vitro and in vivo RDEB data, we herein propose to assess the safety and efficacy of IV gentamicin in RDEB patients with nonsense mutations. We will administer IV gentamicin to 6 RDEB patients daily for 14 days or twice weekly for 3 months and: (i) determine the percentage increase of C7 and AFs in the DEJ of the patients' skin before and after treatment, (ii) determine the post-treatment sustainability of new C7 and AFs in the patients' skin, (iii) determine if this regimen is safe in this patient population, and (iv) determine if it improves the patient's skin lesions and their quality of life. We believe this proposed clinical trial could very well lead to the first novel and non-invasive treatment of RDEB patients carrying nonsense mutations. A successful outcome would have profound therapeutic potential for the 30% of RDEB patients caused by nonsense mutations.